Early Molecular Alterations Research Articles

Early molecular alterations in bronchi and non-small cell lung cancers (NSCLC) from smokers

Early molecular alterations in bronchi and non-small cell lung cancers (NSCLC) from smokers

No mutations found in exons of TP53, H-RAS and K-RAS genes in liver of male Wistar rats submitted to a medium-term chemical carcinogenesis assay

The standard protocol to evaluate the carcinogenic potential of chemicals is the long-term bioassay in rodents, not performed in developing countries due to its high cost and complex operational procedures. Our laboratory has established an alternative medium-term bioassay in Wistar rats, also called DMBDD assay, based on the paradigm initiation/promotion of chemical carcinogenesis. This method was accepted by the Brazilian Environment Agency (IBAMA) as an official source of evidence of carcinogenicity. The aim of this study was to evaluate alterations in exons 5 to 8 of the tumor suppressor gene TP53 and exons 1 and 2 of oncogenes K-RAS and H-RAS in neoplastic and preneoplastic hepatic lesions observed in DMBDD assay. The characterization of these alterations may contribute to the recognition of patterns of damage in critical genes, as well as to suggest mechanisms of action of the compounds tested in the protocol. Sixty male Wistar rats were separated into 3 groups: the first was treated with no chemicals; the second received five initiating agents and the third received initiation followed by phenobarbital. Liver DNA samples (obtained from formalin-fixed and paraffin-embedded tissues after histological analysis) were evaluated by the non-isotopic PCR-SSCP technique. No changes in any analyzed exons were detected by the PCR-SSCP banding pattern in all experimental groups. This result suggests that liver mutations in exons 5 to 8 of TP53 and exons 1 and 2 of H-RAS and K-RAS are not among the early molecular alterations occurring in the hepatic carcinogenesis process induced by the DMBDD protocol in male Wistar rats.

Field defects in progression to adenocarcinoma of the colon and esophagus

A field of defective tissue (field defect) appears to be an early stage in the progression to adenocarcinoma of both the colon and the esophagus. In both cases the field is comprised of cells characterized by resistance to induction of apoptosis, aberrant increased proliferation and genomic instability. Specific mutations and epimutations occur in both types of field. Also there is evidence for aberrant overexpression or underexpression of specific proteins. Most of these early molecular alterations appear to promote either enhanced apoptosis resistance, proliferation, or genomic instability. The molecular alterations that are present early are often, but not always, also present to an even greater degree in the cancer itself. Many of the important events in the development of colonic and esophageal adenocarcinoma first occur in tissues that are histologically neither dysplastic nor malignant. The identification of early molecular defects in morphologically normal appearing tissue is paramount in identifying subjects at high risk for cancer.

Altered regulation of the ET-1 cascade in lambs with increased pulmonary blood flow and pulmonary hypertension.

Plasma concentrations of endothelin-1 (ET-1) are increased in children with congenital heart disease associated with increased pulmonary blood flow. However, the role of ET-1 in the pathophysiology of pulmonary hypertension remains unclear. Preproendothelin-1 gene expression is increased in adults with advanced pulmonary hypertension. To characterize potential early molecular alterations in the ET-1 cascade induced by increased pulmonary blood flow and pulmonary hypertension, fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt). RNase protection assays and Western blot analysis were performed on lung tissue prepared from 4-wk-old shunt lambs and age-matched controls. Endothelin-converting enzyme-1 [the enzyme responsible for the production of active ET-1 from big ET-1, mRNA (411%, p<0.05)] and protein (170%, p<0.05) were increased in lung tissue prepared from shunt lambs, compared with age-matched controls. Endothelin type A receptor (the receptor that mediates vasoconstriction), mRNA (246%, p<0.05), and protein (176%, p<0.05) also were increased in lung tissue prepared from shunt lambs compared with age-matched controls. Conversely, endothelin type B receptor (the receptor that mediates vasodilation), mRNA (46%, p<0.05), and protein (65%, p<0.05) were decreased in shunt lambs. Both the mRNA and protein levels for preproendothelin-were unchanged. Thus we conclude that increased pulmonary blood flow and pulmonary hypertension induce early alterations in the ET-1 cascade that result in increased ET-1 production, increased ET-1-mediated vasoconstriction, and decreased vasodilation. These early alterations in gene expression may contribute to the development of pulmonary hypertension and its associated enhanced pulmonary vascular reactivity.

Frequent Genetic Alterations in Simple Urothelial Hyperplasias of the Bladder in Patients with Papillary Urothelial Carcinoma

In order to understand the origin of bladder cancer, very early urothelial lesions must be investigated in addition to more advanced tumors. Tissue from 31 biopsies of 12 patients with urothelial hyperplasias and simultaneous or consecutive superficial papillary tumors were used to microdissect urothelium from 15- microm sections of biopsies. The biopsies were obtained with the recently developed highly sensitive diagnostic method of 5-aminolevulinic acid-induced fluorescence endoscopy (AFE). Besides flat and papillary urothelial neoplasms, the method of photodynamic diagnostics also detects simple urothelial hyperplasias as fluorescent positive lesions. In addition, 12 fluorescence-positive biopsies showing histologically normal urothelium were investigated. Fluorescence in situ hybridization was done using a dual color staining technique of biotinylated centromeric probes of chromosomes 9 and 17 and digoxigenin-labeled gene-specific P1 probes for chromosomes 9q22 (FACC), 9p21(p16/CDKI2), and 17p13(p53). Ten of 14 hyperplasias (70%) showed deletions of chromosome 9. In 7 out of 8 patients with genetic alterations in the hyperplasias the genetic change was also present in the papillary tumor. Six out of 12 samples of microdissected normal urothelium also showed genetic alterations on chromosome 9. Microdissection of urothelial lesions, obtained during AFE, has led to the first unequivocal documentation of genetic changes in urothelial lesions diagnosed as normal in histopathology. Thus, this technical approach is important to provide insight into the earliest molecular alterations in bladder carcinogenesis.

P53 expression above the basal cell layer in oral mucosa is an early event of malignant transformation and has predictive value for developing oral squamous cell carcinoma

Epithelial dysplasia is usually used to establish the prognosis of oral premalignant lesions. Its assessment, however, is subjective and does not always correctly predict the outcome of the lesions in terms of malignant transformation. Early molecular alteration(s) that dictate the development of cancer should be identified and used to evaluate oral premalignant lesions. In this context, alterations in the expression of p53 were investigated. Thirty-five oral premalignant lesions and 11 carcinomas that developed from them in a period of 16 years were investigated for p53 expression by immunohistochemistry. Normal oral mucosa from healthy individuals and oral benign lesions were used as controls. In benign lesions and normal mucosa, p53 staining, when present, was confined to the basal cell layer. Seven out of 35 (20 per cent) premalignant lesions showed p53 expression clearly above the basal cell layer and six of these (86 per cent) developed carcinomas. Suprabasal p53 expression was found in three lesions with no or mild dysplasia that developed carcinomas. All carcinomas derived from premalignant lesions with p53 suprabasal expression showed p53 expression in neoplastic cells. The combined use of histological parameters (presence of moderate or severe dysplasia) with p53 expression patterns (p53 staining above the basal cell layer) showed the highest sensitivity for the detection of lesions that progressed to carcinoma (91 per cent). When used individually, the p53 expression pattern showed higher specificity than assessment of dysplasia (96 per cent vs. 54 per cent) and higher positive predictive value (86 per cent vs. 44 per cent) for correct prediction of the malignant transformation of the lesions. The results suggest that clear expression of p53 above the basal cell layer is an early event in oral carcinogenesis and an indicator of a developing carcinoma, even preceding morphological tissue alterations. However, since immunohistochemistry cannot always detect changes in p53 expression in lesions preceding carcinoma, p53 immunohistochemical analysis is strongly recommended in conjunction with histological parameters, to increase the sensitivity of detection of cases that will progress to carcinoma. © 1998 John Wiley & Sons, Ltd.

P53 expression above the basal cell layer in oral mucosa is an early event of malignant transformation and has predictive value for developing oral squamous cell carcinoma.

Epithelial dysplasia is usually used to establish the prognosis of oral premalignant lesions. Its assessment, however, is subjective and does not always correctly predict the outcome of the lesions in terms of malignant transformation. Early molecular alteration(s) that dictate the development of cancer should be identified and used to evaluate oral premalignant lesions. In this context, alterations in the expression of p53 were investigated. Thirty-five oral premalignant lesions and 11 carcinomas that developed from them in a period of 16 years were investigated for p53 expression by immunohistochemistry. Normal oral mucosa from healthy individuals and oral benign lesions were used as controls. In benign lesions and normal mucosa, p53 staining, when present, was confined to the basal cell layer. Seven out of 35 (20 per cent) premalignant lesions showed p53 expression clearly above the basal cell layer and six of these (86 per cent) developed carcinomas. Suprabasal p53 expression was found in three lesions with no or mild dysplasia that developed carcinomas. All carcinomas derived from premalignant lesions with p53 suprabasal expression showed p53 expression in neoplastic cells. The combined use of histological parameters (presence of moderate or severe dysplasia) with p53 expression patterns (p53 staining above the basal cell layer) showed the highest sensitivity for the detection of lesions that progressed to carcinoma (91 per cent). When used individually, the p53 expression pattern showed higher specificity than assessment of dysplasia (96 per cent vs. 54 per cent) and higher positive predictive value (86 per cent vs. 44 per cent) for correct prediction of the malignant transformation of the lesions. The results suggest that clear expression of p53 above the basal cell layer is an early event in oral carcinogenesis and an indicator of a developing carcinoma, even preceding morphological tissue alterations. However, since immunohistochemistry cannot always detect changes in p53 expression in lesions preceding carcinoma, p53 immunohistochemical analysis is strongly recommended in conjunction with histological parameters, to increase the sensitivity of detection of cases that will progress to carcinoma.